Anaplastic thyroid cancer (ATC) is characterized by very aggressive growth with undifferentiated features. Recently, it has been reported that the Notch1 signaling pathway, which affects thyrocyte proliferation and differentiation, is inactivated in ATC. However, it remains largely unknown whether using Notch1 activating compounds can be an effective therapeutic strategy in ATC. Therefore, in this study, we aimed to evaluate the drug effects of a potential Notch activator hesperetin on ATC cell.A unique ATC cell line HTh7 was used to evaluate the drug effects of hesperetin. The Notch1 activating function and cell proliferation were evaluated. The mechanism of growth regulation was investigated by the detection of apoptotic markers. The expression levels of thyrocyte-specific genes were quantified for ATC redifferentiation.Upregulated expression of Notch1 and its downstream effectors hairy and enhancer of split 1 (Hes1) and Hes1 related with YRPW motif was observed in hesperetin-treated ATC cells. The enhanced luciferase signal also confirmed the functional activity of hesperetin-induced Notch1 signaling. Hesperetin led to a time- and dose-dependent decrease in ATC cell proliferation. The cell-growth inhibition was mainly caused by apoptosis as evidenced by increased levels of cleaved poly ADP ribose polymerase and cleaved caspase-3 as well as decreased survivin. Additionally, hesperetin induced the expression levels of thyrocyte-specific genes including thyroid transcription factor 1 (TTF1), TTF2, paired box gene 8, thyroid stimulating hormone receptor, and sodium/iodide symporter.Hesperetin activates the Notch1 signaling cascade and suppresses ATC cell proliferation mainly via apoptosis. Hesperetin also induces cell redifferentiation of ATC, which could be useful clinically.